Summary of Work: We have developed a system to examine the consequences of a site-specific double-strand break (DSB) at a YZ junction in dispensable DNA within S. cerevisiae. A galactose- inducible HO-endonuclease cuts a YZ site placed in a plasmid (YZ- CEN) or at various positions within a YAC containing human DNA (YAC12). A persistent, long-lived DSB can lead to G-2 arrest and lethality. Most site-specific breaks in the YACs were rapidly repaired and did not lead to arrest or lethality, nor did a break in the YZ-CEN plasmid when it was rapidly degraded or repaired. By examining different strain backgrounds we have suggested differences in the genetic control(s) responsible for indirect lethality from a persistent DSB. A persistent DSB in a lambda DNA containing YAC (VS8) or the YAC12 derivatives, u8 or u17, did not induce cell cycle arrest or lethality in strain LS20. However, both cell cycle arrest and lethality resulted from a persistent DSB in other strains. We, therefore, examined whether the presence of a high-copy yeast genomic library or a galactose- inducible human testis cDNA library could lead to indirect lethality by a persistent DSB in LS20. Seven yeast genomic fragments and two human cDNAs have been identified that enhance lethality from a persistent DSB. One clone led to the identification of the SIR4 gene in the signaling response. This gene in yeast can affect transcriptional silencing, chromatin organization, aging and double-strand break endjoining. Thus a new role for SIR4 has now been identified. When SIR4 is absent, the unrepairable DSB induced in a dispensable plasmid or artificial chromosome leads to prolonged G2/M arrest as compared to cells containing SIR4. While most of the SIR4 cells eventually divide, there is only a limited number of cell divisions and survival is reduced. The requirement of SIR4 for toleration of an unrepaired DSB suggests that chromatin structure plays an important role in checkpoint adaptation to a persistent DNA lesion.